Currently, various methods of detecting viruses have been used to detect the presence of infectious viruses in blood or blood products, and to identify the presence of viruses in patients with diseases. However, these methods are not always highly sensitive or specific though the sensitivity and the specificity may vary with the type of virus to be detected. Even when they are sensitive and specific enough, they are often expensive and require lengthy procedures as in the culture and isolation of a virus. As a background to the present invention, type C hepatitis (hepatitis C) will be mentioned in detail below.
The causative agent of hepatitis C had long been unknown, but when the gene of the virus was cloned (Science 244: 359-362, 1989) and a diagnostic method by antibody measurement using a recombinant antigen generated based on said gene was developed (Science 244: 362-364, 1989; Japanese Patent Publication (Kohyo) 2 (1990)-500880), it was found that hepatitis C is an infectious disease whose causative agent is hepatitis C virus (HCV) that is transmitted through the blood and blood products as its main route of infection. With the development of the so-called second generation antibody testing method in which a recombinant core antigen and a recombinant NS3 antigen have been added, it is now possible to identify virtually all HCV patients by testing their serum. This has made it possible to eradicate almost all HCV infections transmitted though blood donations in Japan.
However, as for other common viral infections such as by the human immunodeficiency virus (HIV), there is a period of time until the appearance of antibodies after infection, or the so-called window period in which a virus is unidentifiable by existing testing methods. This means that the risk of secondary infection is still present, due to blood-borne components that cannot be identified by antibody testing methods, in areas where blood-selling is legal or in some regions of Japan. The antibody testing method also has a drawback in that it cannot distinguish a person who has recuperated from an infection and a person who is in the active stage of infection because of its principle of testing.
Interferon (IFN) is currently used for the treatment of hepatitis C. Some researchers insist, however, that the efficacy of the therapy can be evaluated by only measuring the antibody titer of HCV because the titer declines 6 months after elimination of HCV by IFN. However, since the antibody titer starts to decline only after the reduction of antigen stimulation or several months after the elimination of antigen, it is impossible to determine whether IFN administration resulted in the elimination of HCV, at a desired timing and accuracy, by the antibody testing alone. Hence, in order to monitor the therapy, it is necessary to detect HCV per se in addition to the HCV antibody.
It was difficult to establish a method of directly detecting the virus particle (virus antigen) of HCV because blood levels of the virus are very low as compared to other viruses such as hepatitis B virus (HBV) and because the virus cannot be propagated in vitro or using an animal etc. as a host. Therefore, instead of detecting the virus antigen, methods of detecting the genomic RNA of the virus were developed such as the polymerase chain reaction (PCR) method (Science 230: 1350-1354, 1985) and the branched-chain DNA probe method. But, the method of detecting viral genomes have several problems when compared to the method of detecting virus antigens.
First, it has been pointed out that since the substance to be detected is RNA that is not very stable during storage, the procedure of freezing and thawing of serum may cause a reduction in the measured value. Thus, the serum samples to be tested must be stored more carefully than when they are used in other assay methods. Utmost care must also be taken in transportation of the samples.
Although the testing methods that involve the use of a PCR method are the most sensitive for detecting gene fragments, they have problems in that: reverse transcription from a genomic RNA to a template DNA is often accompanied by losses, which therefor requires great skills to obtain an accurate quantitative value, and: since amplification is an important principle in the methods, a high incidence of false-positives may occur in case of contamination, and thus the processing of a large volume of samples at one time is impossible. Furthermore, even those methods which are postulated to be a simple procedure take 2 hours or more for pretreatment of samples and are complicated since repeated procedures of centrifugation and the like are required. In addition, such complicated procedures lead to increased chances of contamination and thereby increased chances of obtaining false-positive results. On the other hand, the branched-DNA probe method is low in detection sensitivity and besides takes about 20 hours before obtaining test results (Igaku to Yakugaku [Medicine and Pharmacology] 31: 961-970, 1994), and hence the method leaves much to be desired in terms of sensitivity and processing time.
In order to solve the above-mentioned problems associated with the methods of detecting viral genomes, methods were developed that involve the direct detection of a virus antigen. As shown in Japanese Unexamined Patent Publication (Kokai) No. 8 (1996)-29427, a method was developed that detects the core antigen of HCV in the serum using monoclonal antibody specific for the core antigen. As has been reported in Tanaka et al., Journal of Hepatology 23: 742-745, 1995, and Fujino et al., Igaku to Yakugaku [Medicine and Pharmacology] 36: 1065-1070, 1996, methods of detecting the core antigen in the serum have been shown to have a clinical usefulness as do the above-mentioned methods of detecting the viral genome. However, there are still several major problems that need be solved as in the methods of detecting the viral genome.
One such problem is that the sensitivity, compared to the PCR method, is so low that it cannot be used as a final test method of serum screening. Tanaka et al., Journal of Hepatology 23: 742-745, 1995, indicated that the detection limit is 104-105 copies/ml of HCV RNA. Fujino et al., Igaku to Yakugaku [Medicine and Pharmacology] 36: 1065-1070, 1996, reported that the method has shown a positive rate of 67% on 102 sera of the patients before treatment with chronic hepatitis C who were found to be RNA positive by the most sensitive detection method of CRT (competitive reverse transcription)-PCR method. That is, in terms of sensitivity, the method lags far behind the most sensitive CRT-PCR method.
Furthermore, the complicated procedure of treating samples for measurement, and the long time it takes, pose problems when it is used in screening. Thus, the method requires a multi-step procedure for sample (serum) treatment comprising: a polyethylene glycol treatment (4° C., 1 hr) for the concentration of virus particles and the removal of serum components; centrifugation (15 min); the removal of supernatants; urea treatment; the alkali treatment (37° C., 30 min); the addition of the neutralizing agent and the like. In addition, the process of dispersing, with urea, the precipitate having an increased viscosity due to the PEG treatment requires great skill. In order to obtain a reproducible result, therefore, great skill is required and, besides, a minimum of 2 hours of treatment is necessary. Furthermore, such processes as centrifugation, supernatant removal, etc. are not amenable to automation and render the simultaneous treatment of a large number of samples very difficult. Thus, from a viewpoint of ease of handling as well, the method is not suited for applications that require the treatment of a large volume of samples as in screening tests.
On the other hand, the virus antigen detection system is superior to the highly sensitive PCR method in the following points. Thus, it is very tolerant to contamination because it involves no procedure of excessive amplification in the detection step. Furthermore, since it is intended to detect antigen protein that is relatively stable instead of poorly stable RNA, it requires no excessive care in the storage of samples, it does not require special equipment such as the deep freezer that is needed for samples to be detected by PCR, and the transportation of the samples is also easier.
These features are suitable for applications in which a large number of samples is measured as in the blood industry or health checkup testing. However, because the disclosed method of detecting the core antigen, as indicated above, is not amenable to automation and is low in sensitivity so that it cannot be a gold standard in applications that require high sensitivity such as in the blood industry, it cannot be applied to tests that handle a large number of samples such as screening, and cannot make the best use of its advantageous features over the PCR method. Furthermore, clinically useful assay methods must always face the challenges of sensitivity, specificity, reproducibility, ease of handling, and low cost, and sustained efforts are needed to satisfy these challenges as much as possible. With regard to detection of virus antigens other than HCV, especially for use in screening handling a large number of samples, there are many methods that are not put into practical use because they are low in sensitivity, as compared to the PCR method, or the desired antigen could not be fully exposed.